CA1179372A

CA1179372A - 1-branched-alkyl-3-(2-haloethy)-3-nitrosoureas as novel antitumor agents

Info

Publication number: CA1179372A
Application number: CA000347028A
Authority: CA
Inventors: Koloman Laki
Original assignee: National Foundation for Cancer Research
Current assignee: National Foundation for Cancer Research
Priority date: 1979-08-08
Filing date: 1980-03-05
Publication date: 1984-12-11
Also published as: CH643237A5; FR2465716B1; NO151545C; US4423076A; GB2056450A; DE3020498A1; AU532391B2; DK281980A; GB2056450B; HU182184B; NL8001602A; NO802348L; NO151545B; FR2465716A1; JPS5653647A; SE8005574L; IT1149819B; IT8024040A0; AU5703880A

Abstract

ABSTRACT OF THE DISCLOSURE
A process for preparing a compound of the general formula:

wherein, R is neopentyl, neohexyl, isopentyl or isobutyl and Hal is chlorine or fluorine, the process comprising, nitrosolating a compound of the general formula:

Description

~ 17937~

~ rhe L)resent invention relates to novel nitrosourea derivatives useEul for their antitumor activity.
In the past decade, the nitrosoureas have gained acceptance as potent antitumor agents (Johnston, _t al., J. Med. Chem., 14:600 (1971)). The accepted mode of action appears to be through the release of isocyanate ln vivo.
The -two compounds most frequently used clinically are l-cyclohexyl-3-(Chloroethyl)-3-nitrosourea(CCNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea(BCNU) which release in vivo an isocyanate derived from the unnitrosated side of the molecule, and an alkylating-agent from the other side.
Numerous studies have been directed toward the meta-bolic products produced ln vlvo and ln itro in aqueous me~dicl, which mainly consist of 2-chloroethanol, vinyl chloride, acetaldehyde, anddichloroethane (Johnston, ~t al., J. Med.
Chem., 18:634 (1975)). It is also known that the N~nitroso-N-alkyl ureido portion of the molecule alkylates DNA
(deoxyribonucleic acid) in vivo and in vitro (Frei, et al., Biochem. J., 174:1031 ~1978)). In fact, it has been shown that the carcinogenic effectiveness of agents such as N-methyl-N-nitrosourea correlate with the extent of alkylation of the guanine moiety in DNA of target tissues at the C~6 atom.
Alkylation of DNA occurs within an hour after administration of the nitrosourea, and the half-life of the alkylated products is about 24 to 48 ho~rs (Reed, et al., Cancer Res., 35:568 (1975)). The study indicated that low doses of nitrosoureas pose only a small threat as mutagens, and hence, are not significantly carcinogenic. From this it may be postulated that the more transglutaminase-specific ,'r ~ ~937~

(see below) tlle isocyanate resulting from decomposition of the nitrosourea, the lower the required dose, resulting in a reduced risk of carcinogenesis Erom the antitumor agent.
It has recently been showrl that a number of isocyan-a-tes are potent inhibitors of the enzyme transglutaminase (Gross, et al., J. Biol. Chem., 250:7693 (l975)), a calcium-dependent enzyme which cataly~es the lysine-glutamine cross-linking of certain proteins present on neoplastic cell surfaces.
This enzyme has been implicated in the uncontrolled prolifera-tion of cancer cells (Yancey and ~aki, Ann. N.Y. Acad. Sci., 202:344 (1972)). It has been proposed that these cross:linked proteins form an extracellular coating causing the cell to be unrecognized by the cellular immune system, thus pre~ent:ing normal destruction of foreign neoplastic -tissue. The enzyme is fairl~ speciEic toward glu-tamine residues ~s su~s-trates, and isocyanates resembling these residues have been ~ound to be the mos-t effective inhibitors (Gross, et al., ~. Biol.
Chem., 250:7693 (1975)).
The structure oE the active site of the -transgluta-minase has been found to contain the pentapeptide sequence --Tyr-Gly-Gln-Cys-Trp-- and has the shape of a pocket approximately 5 x 5 Angstroms tFo1k and Cole, J. ~iol. Chem., 241:3238 (1966)).
According -to the hypothesis upon which the invention is based, a superior inhibitor of transglutaminase would have, in view of the sequence and size indicated above, hydrophobic moieties directed away from, but in proximity to, the pocket at the active site. Two possible inhibitors meeting these criteria are neopen-tyl isocyanate (I) and neohexyl isocyanate (II) which can be seen to resemble 1 ~7g3P~2 gluta111ine (III) in size:

Ctl3 CH3 CH3-f-CE12-N-C=OC113~ -C~12-CH2-N=C=O

Ctl3 CH3 (I) (~I) 1 ~12 H-C-CH -CH - J o j=0 NH2 0~1 (III) ~he hypothesis oE the invention is supportecl by the -fact that t-butyl groups, or other hydrophobic groups, attachecl to the be-ta- or gamma-carbon of glutamic acid produce superior substrates for transglutaminase (Gross and Folk, J. Biol. Chem., 248:130 (1973)). Ester analogs oE these compounds are also substrates ~Gross and Folk, J. Biol. Chem., 249:3021 (l974)).
It has been shown that isocyanates inhibi-t the transglutaminase through alkyl thiocarbamate ester formation through the single active site sulfhydryl group (Gross, et al., J. Biol. Chem., 250:7693 (1975)):

I 179~72 H O
l 11 R-SH + R'-N=C-O ~R'-N-C-SR

Bis-(neopentyl)-N-nitrosourea (IV) CH O CH

CH3-f-CH2-W-C-NH-CH2-f-CH3 (IV) was synthesized as a compound which woulcl release neopentyl isocyanate (I) ln _vo resulting in inhibition of trans~
glutaminase. However, the limited solubility of this com-pound prevented its clinical evaluation.
Recent studies have shown -that the activity of nitrosoureas is markedly enhanced by the presence of the

2-chloroethyl group on the nitrosated side of the compound (Montgomery, Clncer Treat. Rep., 60:651 (1976); Johnston, et al., J. Med. Chem., 9:892 (1966); Farmer, et al., J. Med.
Chem., 21:514 (1978)). Since the 2-chloroethyl group also enhances solubility, it became the group of choice at the

3-position in this study.
According to an aspect of this invention there is provided a process for preparing a compound of the general formula:
Il R- NH- C- N- CH2- CH2- Hal (A) N=O

wherein R is selected from the group consisting of neopentyl, neohexyl, isopentyl and isobutyl and Hal is selected , I
" .

~ ~7937~

from -the group consisting oE chlorine and fluorine, the process comprisiny nitrosolating a compound of the general formula:

R- NH- C -NH- CH2- CH2- Hal with nitrous aci.d.
Compounds having the formula (A) have an'itumor activ-itY and are novel. In accordance with another aspect of the invention such compounds are provided whenever produced by the process aspect of the invention, or by an obvious chemical equivalent thereof.
The following compounds of the invention were synthesized as described in the Experimental section:

o R- NH- C - N- CH2 CH2 Hal N=O

,)o V, R = Neopentyl VI, R = Neohexyl VII, R = Isopentyl VIII, R = Isobutyl Hal = Cl or F

~ 179~72 Chemical studies revealed that the neopentyl and neohexyl isocyanates are remarkahly stable in water-acetone (1:3) at 57 C without substantial amounts being hydroly~ed even after a three-day period at pH 6Ø Both have half-lives of greater than 30 minutes at physiological pH and tempera-ture.
Thus, the branched alkyl groups of the compounds of the invention were selected using the criterion that they should be substrates for, and thus be selective inhibitors of, transglutaminase. As noted above, the work oE Montgomery discloses a single l-branched-alkyl-3-~2-chloroethyl)-3 nitrosourea, namely l-(l-me-thyl-hexyl)-3-(2-chloroethyl)-3-ni-trosourea (IX):

cl~3cf~2c~2cH2c~l2- fH~ NH- C- N- CH2CH2~ Cl (IX) While this compound is a l-branched-alkyl-3-(2-chloroethyl)-3-nitrosourea, it does not fulfill the steric criteria outlined above for optimal inhibitory activi-ty.
It is not desired to limit the invention by the theoretical considerations presented herein, and the above discussion is included merely for purposes of background discussion.
EXPERIMENTAL SECTION
Melting points were determined using a Thomas Hoover capillary melting point appara-tus and are uncorrected.

InErarecl (IR) spectra were obtainecl using a Perkin Elmer 397 spectrophotometer, and NMR (nuclear magnetic resonance) spectra were taken on a Br~ker ~P80DS system with tetramethyl-silane as an internal standard. Elemental analyses were carried out by Calbraith Laboratories, Knoxville, Tennessee.
Butyl acetyl chloride and 3,3 dime-thyl-l-butallol were obtained from Aldrich Chemical Co. and were used wlthout further purification as a result of sa-tisfactory NMR analysis.

1-Neopentyl-3-(2-Chloroethyl~ Urea. Neopentylamine (8.7 g, 0.1 mol) was dissolved in 50 ml of anhydrous diethyl ether and cooled to 5C. 2-Chloroethyl isocyanate (10.5 g, ().1 mol) dissolved in an additional 50 ml of ether was aclded over :30 minutes while maintaining a tempera-ture of less than 1() C.
Stirring continued for an addi-tional hour, ancl ~he cold mixture was filtered and washecl with chilled ether. The compound was dried in a desiccator over sodium hydroxide overnight giving 15.4 g (80%) as a whi-te powder, MP (meltiny point) 90-1C (decomposes). Elemental analysis showed the following:
Calculated Found Carbon 49.97% 49.87%
Hydrogen 8.87% 8.89%
Nitroyen 14.52% 14.54%
Chlorine 18.47~ 18.40%
IR analysis showed bands at 1630/cm (C=O) and 1535/cm (N-C=O). NMR showed a singlet (9H) at 0.8 ppm and a mutliplet (6H) at 3.4 ppm.

1 1 7~372 l-Neopentyl-3-~2-Chloroethyl)-3-Nitr~sourea (V). The entire _ __ _ _ _ yield of l-neopentyl-3-(2-chloroethyl~ urea (0.08 mol) was dissolved in 120 ml of concentrated HCl:ethanol, 2:1 at 5 C
in a 500 ml round bo-ttom flask equipped with magnetic stirrer. Sodium nitrite (5.5 g, 0.08 mol) was dissolved in 30 ml of water and added to the solution over a ten-minute period. Stirring continued for 2 hrs., and the yellow, crystalline precipita-te was filtered and washed with five 100 ml portions of chilled distilled water. The product was dried in vacuo for 18 hrs. giving 15 g (84%~ which melted with decomposition at 52-3C. IR analysis showed peaks at 1730/cm (C=O~ and 1520/cm (C-N-H). NMR showed a singlet (9EI) at 1 ppm, a doublet (2H, J = 6.5 Hz) at 3.3 ppm, a triplet (2H, J -- 6.5 llz) at 3.6 ppm, and a triple-t (2H, J = 6.5 Hz) at 4.2 ppm.

1-Neohexyl-3 ~ . A 5 g quantity of 3,3-dimethyl butylamine (0.5 mol) was dissolved in 30 ml of anhydrous diethyl ether and stirred on an ice bath.
2-Chloroethyl isocyanate (5.25 g, 0.5 mol) dissolved in 15 ml of ether was added maintaining a temperature of 10 C or less, and stirring continued for 2 hrs. Fil-tering the product and washing with four 5 ml portions of chilled ether afforded 6.2 g (60%) which melted at 84C (decomposes). IR
analysis showed peaks at 1625/cm (C-O) and ]580/cm (N-C=O).
NMR: singlet (9H) at 0.9 ppm, multiplet (2H) at 1.3 ppm, multiplet (2H) at 3.2 ppm, multiplet (4H) at 3.5 ppm, singlet (lH) at 5.5 ppm, and singlet ~lH) at 5.7 ppm.

`1~

1 ~79372 _Neohexyl-3-(2-Chloroethyl)-3--Nitrosourea (VI). A 2.07 g (10 mmol) quantity of 1-neohexyl-3-~2-chloroethyl) urea was dissolved in 15 ml of concentrated HCl:ethanol, 2:1, at 10 C, and 690 mg (10 mmol) sodium nitrite dissolved in 3 ml oE water was added. Stirring at 0.5C continued Eor 1.5 hrs.
and the precipita-te was washed with four 25 ml portions of distilled water. After drying in vacuo overnight, 1.7 g (74%) of the product was obtained which meltecl at 43.0-43.5C (decomposes). IR analysis showed peaks at 17C5/cm (C=O) and 1525/cm (C-N=O). NMR: singlet (9H) at 0.9 ppm, mul-tiplet (2H) at 1.3 ppm, symmetrical multiplet (6H) at 3.8 ppm. No evidence of isomeric ma-terial was detected.

l-Isopentyl-3-(2-Chloroethyl) Urea. Isopentylamine (8.7 c3, 0.1 mol) was dissolved in 100 ml of anhydrous ether and cooled to less -than 10C. 2-Chloroethyl isocyana-te (10.5 cJ, 0.1 mol) was dissolved in 20 ml of ether and added to -the rapidly stirring reaction mixture at less than 10C. Stirring was continued for 1 hr. longer and the precipitate was filtered and washed with four 20 ml portions of chilled ether.
After drying in vacuo overnight, 16.5 g (85~) of product was obtained as a white powder, MP 58-9C. IR analysis showed peaks at 1620/cm (C=O) and 1575/cm (N-C=O). NMR showed a doublet (6H, J = 7 Hz) at 0.9 ppm, a triplet (2H,J = 7 Hz) at 1.4 ppm, a multiplet (lH) at 1.8 ppm, a multiplet (lH) at 3.2 ppm, a multiplet (4H) at 3.6 ppm, a singlet (lH) at 5 7 ppm, and a singlet (lH) at 6.0 ppm.

l-Isopentyl-3-(2-Chloroethyl)-3-Nitrosourea (VII). A 1.92 g (10 mmol) quantity of 1-isopentyl-3-(2-chloroethyl~ urea ~.

was dissolved in 15 ml o~ HCl:ethonal, 2:1, and cooled to 5C. Sodium nitrite (690 mg, 10 mrnol) dissolved in 3 ml of water was added in portions with stirring, after which stirring was continued for an additional 2 hrs. The mixture was filtered and 50 ml of water was added. Extracting with two 20 ml portions of ethyl acetate Eollowed by evaporation of the solvent afforded the product as an oil which did not solidify on standing. Similar resul-ts were obtained using 98% formic acid as the solvent. IR analysis showed peaks at 1705/cm (C=O) and 1525/cm (N-C=O~. NMR analysis showed a doublet (6H, J = 7 Hz) at 0.9 ppm, a triplet (2H, J = 7 Hz) at 1.4 ppm, a multiplet (lH) at 1.8 ppm, a multiple-t (6H) at 3.8 ppm, and a singlet (lH) a-t 7.4 ppm.

l-Isobut~l-3-(2-Chlor ~ ~rea. Isobu-tylamine ~7.3 ~, 0.1 mol) was dissolved in 20 ml of die-thyl ether and cooled to less than 5 C. Chloroethyl isocyana-te (10.5 9, 0.1 mol) dissolved in 15 ml of ether was added maintaining a tempera-ture between 0 and 5 C with vigorous stirring. Stirring was continued for an additional 2 hrs. the mixture was cooled to -5C, and the product (4.1 g, 46~) was filtered and dried in vacuo for 18 hrs. over KOH, MP 79.5 C (decomposes).
IR analysis showed peaks at 1630/cm (C=O) and 1585/cm (N-C=O).
NMR: Doublet at 0.9 ppm (J = 6.4 Hz), multiplet (lH) a-t 2.1 ppm, quartet (2H) at 2.9 ppm (J = 6.4 Hz), multiplet (4H) at 3.5 ppm.

l-Isobutyl-3-(2-Chloroethyl)-3-Nitrosourea (VIII). A 2 g (11.2 mmol) quantity of 1-isobutyl-3-(2-chloroethyl) urea was dissolved in 25 ml of concentrated HCl:e-thanol, 2:1, .
.-1 1~9372 and cooled to S C. A solution of 773 mg ~11.2 mmol) o~ sodium nitrite in 5 ml of water was added in portions and the mixture was stirred at this temperature for 2 hrs. The crystalline yellow solid was filtered and dried in vacuo overnight giving 1.6 g (70~) oE the product, MP 51C
(decomposes). IR analysis showed peaks at 1705~cm (C=O) and 1525/cm (C-N=O). NMR: Doublet (6H, J = 6.8 Hz) at 0.9 ppm, multiplet (1~l) at 2.0 ppm, multiplet (6H) at 3.7 ppm.

1-Branched-alkyl-3-(2-fluoroethyl)-3-nitrosoureas. Syntheses corresponding to those described above may be performed using the 2-fluoroethyl compounds to produce the l-branched-alkyl-3-(2-fluoroethyl)-3-nitrosoureas.

CLINICAL STUDIES
Exper1ment I
A single dose of l-neopentyl-3-(~-chloroethyl)-3-nitrosourea (NCNU) was injected intraperitoneally (ip) in CDF1 mice which had been implanted ip two days earlier with approximately 10 x 105 murine leukemia L1210 tumor cells.
The NCNU was dissoIved in Emulphor EL-620 (polyoxyethylated;
GAF Corporation, New York, N.Y.) and raised to the desired volume with 0.85~ sodium chloride. The increase in median lifespan (ILS) of the test animals above control (untreated) animals is presented in Table I.

* A Trade Mark TABLE I
Effects of Single-Dose Treatment with NCNU Against _ 'l'wo-Day Murine Leukemia L1210 Dose (mg/kg) '~ ILS
_ 100 275 ~
275 +

37.S

Experiment II
The s-tudy of Experiment I was conducted using approximately 5 x 105 murine leukemia P388 tumor c~lls (0.1 ml of a 1:100 dilu-tion). Results are presented in Table II.

TABLE II
.
Effects of Single-Dose Treatment with NCNU Against Two-Day Murine Leukemia P388 Dose (mg/Mouse) % ILS
-2.0 483 +
1.0 91.7 0.5 41.5 , The plus sign indicates that the animals have not yet all died and the experiments are con-tinuing.

;

r 1 ~937~

Experiment III
The eEfect o:E NCNU was tested against Yancey's lymphocytic leukemia (YLL) which had been implanted sub-cutaneously (sc) two days before injection of the drug. The single injection of 0.1 ml of a solution of one spleen of a YLL bearing mouse homogenized in 10 ml Locke's Solution was given ei-ther intraperi-toneally or subcutaneously. Table III presents the increase in median li~e span of test animals over control animals.

TABLE III
Effects of Single-Dose Treatment with NCNU Against Two-Day Yancey ~ ~ kemia Dose Route of Injection % ILS

2.0 mg/mouse ip 264 +
1.0 mg/mouse ip 64.3 0.5 mg/mouse ip 14.3 25 mg/kg ip 13.3 50 mg/kg ip 60.0 75 mg/kg ip 66.6 2.0 mg/mouse sc 78.0 1.0 mg/mouse sc 64.3 0.5 mg/mouse sc 35.7 25 mg/kg sc 6.6 50 mg/kg sc 60.0 75 mg/kg sc 73.3 1.0 mg/mouse ip 33.3 0.5 mg/mouse ip 6.7 1.0 mg/mouse sc 20.0 0.5 mg/mouse sc 13.3 The plus sign indicates that the animals have not yet all died and -the experiments are continuing.

1 17g3~

E:xpe~iment LV
The study oE Experiment III was conducted with NCNU treatment beginniny at 2 or 7 days after implantation of YLI. tumors. NCNU injec-tion was ip or sc on a schedule of one time per week for 4 weeks, twice per week for 4 weeks, or a single dose, Results are provided in Table IV.

TABLE IV
Effects of NCNU Against Yancey's Lymphocytic Leukemia Day After Implantation Treatment Route of Injection Dose_Was Begun In~ectlon Schedule % ILS
1.0 mg/mouse 7ip once 66.6 0.5 mg/mouse 7ip once 6.7 1.0 mg/mouse 7sc once 66.6 0.5 mg/mouse 7sc once 6.7 2.0 mg/mouse 2ip lx/wk ~or 4 wks 14.3 1.0 my/mouse 2 ip " 178.6 0.5 mg/mouse 2 ip " 28.6 75 mg/kg 2 ip " 100 +
50 mg/kg 2 ip i, 100 +
25 my/kg 2 ip " 100 +
2.0 mg/mouse 2 sc " 264 +
1.0 mg/mouse 2 sc " 207.1 +
0.5 mg/mouse 2 sc " 114.3 +
75 mg/ky 2 sc " 100 +
50 mg/kg 2 sc " 100 +
25 mg/kg 2 sc " 100 +
75 mg/kg 2 ip 2x/wk Eor 4 wks 66.6 50 mg/kg 2 ip " lO0 +
25 mg/kg 2 ip " lO0 +
75 mg/kg 2 sc " 0 50 mg/kg 2 sc " lO0 -~
25 mg/kg 2 sc " 100 +

The plus sign indicates that the animals have no-t yet all died and the experiments are continuing.

~-I ~L 793~

Experiment_V
Toxicity s-tudies were conducted by injecting a single dose of NCNU ip. Survival data is set forth in Table V.

TABI.E V
Toxicity of NCNU in CDFl Normal Mice Dose Dea-ths (%) 1.0 mg/mouse 0 2.0 mg/mouse 60 at day 13, others survived 104.0 mg/mouse 100 at day 10 8.0 mg/mouse 100 at day 8 16.0 mg/mouse 100 at day 4 20.0 mg/mouse lQ0 at day 1 32.0 mg/mouse 100 at day 1 100 mg/kg 100 at day 13 150 mg/kg 100 at day 11 200 mg/kcJ 100 at day 9 225 mg/kg 100 at day 9 250 mg/kg 100 at day 8 500 mc3/kg 100 at day 5 750 mg/kg 100 a-t day 2 1000 mg/kg 100 at day 1 PHARMACEUTICAL COMPOSITIONS
The compounds of this invention can be employed in useful pharmaceutical compositions in such dosage forms as tablets, capsules, powder packets, liquid solutions, suspen-sions or elixirs for oral administration; liquid for parenteral use, and in certaln cases, suspensions for parenteral use.
In such compositions, the active ingredient will ordinarily be present in an amount of at least 0.5~ by weight based on the total weight of the composition and not more than 95~ by weight.
Besides the active ingredient compound of this invention, the antitumor composition can contain a solid or liquid non-toxic pharmaceutical carrier for the active ~ ,.

~ 17937~

in~redient.
The capsules, tablets, and powders will generally constitute from about 1 to about 95~ and preferably from about 5 to 90~ by weight of active ingredient. These dosage forms preferably contain from about 5 milliqrams to about 500 milligrams of active ingredient, with about 7 milligrams to about 250 milligrams most preerred.
The pharmaceutical carrier can be a sterile liquid such as water, or a suitable oil, including those of petrol, animal, or ve~etable oil of synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. In general, water, saline, aqueous dex-trose (glucose) and related sugar solutions and glycols such as propy:lene glycol and polyethylene glycols are preferred liquicl carriers particularly for injectible solutions. Sterile in~ectible solutions will ordinarily con-tain from about 0.5 -to about 25~ and preferably about 1 to about 10~ by weight of the active ingredient.
Oral administration can be in a suitable suspension or syrup, in which the active ingredient ordinarily will constitute from about 0.7 to about 10~ and preferably about 1 to about 5% by weight. The pharmaceutical carrier in the composition can be an aqueous vehicle such as an aromatic water, a syrup, or a pharmaceutical mucilage.
Suitable pharmaceutica~ carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, a well-known reference text in this field.
The following examples will further illustrate the preparation of pharmaceutical compositions of the invention.

1 ~9372 EXAMPL~ A
A large number of unit capsules were prepared by filling standard two-piece hard gelatin capsules each with 250 milligrams of powdered 1-neopentyl-3-(2-chloro~thyl)-3-nitrosourea, 110 milligrams of lactose, 32 milligrams of talc, and 8 milligrams stearate.

EXAMPLE B
A mixture of l-neopentyl-3-(2-fluoroethyl)-3-nitrosourea in soybean oil was prepared and injected by meansof a positive displacement pump into gelatin to form soEt gelatin capsules containing 35 milligrams of the active ingredient. The capsules were washed in petrole-lm ether and dried~

EXAMPLE C
A large number of tablets were prepared by conventional procedures so tha-t the dosage unit was 100 milligrams of active ingredient, 7 milligrams of ethyl cellulose, 0.2 milligrams of colloidal silicon dioxide, 7 milligrams of magnesium stearate, 11 milligrams of microcrystalline cellu-lose, 11 milligrams of cornstarch and 98.8 milligrams oE lac-tose. Appropriate coatings may be applied to increase palata-bility or delay absorption~

EXAMPLE D
A parenteral composition suitable for administration by injection was prepared by stirring 1.5% by weigh-t of l-isopentyl-3-(2-chloroethyl)-3-nitrosourea in 10% by volume propylene glycol and water. The solution was sterili~ed by 1 ~7~7~

filtration.

EXAMPL_ _ An aqueous suspension was prepared for oral adminis-tration so that each 5 milliliters contained 50 milligrams of finely divided l-neohexyl-3-(2-chloroethyl)-3-nitrosourea, 500 milligrams of acacia, S milligrams o-f sodium benzoate, 1.0 gram of sorbi-tol solution, U.S.P., 5 milligrams of sodium saccharin, and ~.025 milliliters of vanilla tincture.

EXAMPLE F
A parenteral composition suitable for adminis-tration by injec-tion was prepared by dissolvjng 1% by weigllt of l-neopen-tyl-3-(2-chloroethyl)-3-nitrosourea in sodium chloride injection U.S.P. XV and adjusting the pH of the solu-tion to between 6 and 7. The solution was sterilized by filtration.

A wide variety of compositions can be prepared by substituting other compounds embraced by this invention for the specific compounds named in Examples A-F above and substituting other suitable pharmaceutical carriers described in "Remington's Pharmaceutical Sciences".
The compounds of this invention can be administered in the treatment of any of the various forms of cancer by any means that effects contact of -the active ingredient compound with the site of action in the body of a warm-blooded animal. For example, administration can be parenteral, i.e., subcutaneous, intravenous, intramuscular, or intra-peritoneal; alternatively or concurrently, administration can be by the oral route.

al793~

For the purpose of this disclosure, a warm-blooded animal is a member of the animal kingdom possessed of a homeostatic mechanism and includes mammals ancl birds.
The dosage administered is dependent on the age, health, and wei~ht of the recipient, the extent of disease, kind of concuxrent treatment, frequency of treatment and the effect desired. Usually, a daily dosage of active ingredient compound can be from about 0.1 to lS0 milligrams per kilogram of body weight. Ordinarily, from 2.0 to 75, and preferably 10 to 50 milligrams per day administered in one or more doses daily is effective to obtain the desired results.

HUMA STUDIES
Patients having advanced stages of various forms oE
cancer were treated by subcutaneous injection of two 25 mg/kg doses of NCNU in water or by intravenous administration of 1 milligram NCNU/ml 0.9% saline to provide a total daily dose of 50 mg/kg; a full clinical examination was carried out prior to and after administration. Tumor grow-th was measured where possible by palpitation, x-rays and photo-graphy.
Following treatment patients showed marked clinical improvement within several days. Inflammation gradually subsided and tumor size frequently decreased.
The following types of cancer in addition to others, may be treated with compounds of the invention: tl) undif-ferentiated carcinoma of the (R) ]cidney with metastases, (2) adenocarcinoma of the colon with liver metastases, (3) intraduct carcinoma of the breast (stage IV) with bone metastases, (4) recurrent melanoma, (5) adenocarinoma of the .~ ,'~

7 ~793~2 breast wlth brain metastases and (6) squamous cell carcinoma of the lung.
The above clinical s-tudies establish that the l-branched-alkyl-3-(2-haloethyl)-3-nitrosourea compounds oE
the invention are useful in the treatment of various forms of cancer. Continuous intravenous administration inhibits tumor activi-ty and frequently leads to a general remission of the disease.
The mode of action of the compounds of the invention is, at this point, unclear. However, the empirical observation that cells stop prolifera-tion when exposed to these compounds is sufficient to warrant their use in thq treatment oE such serious, heretofore untreatable, and often fatal diseases, such as cancer.

. ,,, .~

l 1~937,'~

SUPPLEMENTARY DISCLOSURE
In the principal disclosure, there are described certain specific l-branched-alkyl-3-(2-haloethyl)-3-nitroso-ureas havilg antitumor activity and the preparation thereof.
It has now been found that a wider group of compounds than the specific ones disclosed in the principal disclosure have antitun,or activity.
Compounds in accordance with the following criteria have been found to have antitumor activity:

1. The compounds should contain an alkyl isocyanate component of a nitrosourea. It is known that the nitrosourea, as in or example BCNU and CCNU, dissociates ln vivo and in v _ o to yield the isocyanate and diazotic acid ractions. The isocyanate thus produced then acts as the "active site" inhi-bitor for the transglutaminase as discussed in the main dis-closure.
2. At present it is believed that the diazotic acid component should be the same as that for BCNU and CCNU.
3. The compounds should contain an alkyl group of preferably 3 to 8 carbon atoms attached to the isocyanate group.
~roups with more than 10 carbon atoms present solubility pro-blems while methyl and ethyl isocyanate would generally be too toxic (carcinogenic).

4. To be effective, the Cl of the above noted alkyl group should be primary, i.e. with no branching or substituents, since it is '-nown that such compounds, with tertiary and probably also with secondary, Cls would be ineffective becuase of stearic hindrances which would preclude access by the alkyl isocyanate to the active site of the enzyme. Preferably, the alkyl group should be branched, e.~. with method groups at the C2 or C3 posi-tions. However, C4 substituted or branched alkyls are believed likely to be less effective inhibitors because of steric _ ~, _ ~ 1 ~93~72 hindrances. In summary it can therefore be said that branch-ing on the first carbon adjacent to the isocyanate group is in-effective, that on the second carbon, highly effective and that decreasing activity would result as the branching moves out along the chain.

5. In light of the substrate specificity of trans-glutaminase the above notea alkyl group should preferably conform as closely as practicable to a glutamine residue. Howe~er, it has been demonstrated that isocyanate containing an alkyl chain 8 carbons long is still a good inhibitor of transglutaminase.
From the above criteria compounds of general formula X have antitumor activity by lnhibiting transglutaminase.

R NH ~ I--CH2 CH2 al X
NO

wherein ~ is an alkyl group of 3 to 8 carbon atoms with no branch-ing on the Cl and Hal is chlorine or fluorine. Those compounds form one aspect of the invention of this supplementary disclo-sure, whenever prepared by the process aspect hereof defined below.
A preferred embodiment of compounds of formula X
embodies branching on the C2 or C3 of the R group; and a further preferred embodiment of X is ~here the R group sterically conforms to a glutamine residue.
Compounds of the formula X are prepared, in accordance with another aspect of the invention of this supplementary disclosure, by nitrosolating a compound of the formula XI with nitrous acid O
R - NH - C - HN - CH2 - CH~ - Hal XI

~ ~ -- `2 ;L --~J

Claims

The embodiments of the lnvention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing a compound of the general formula:

wherein R is selected from the group consisting of neopentyl, neohexyl, isopentyl and isobutyl and Hal is selected from the group consisting of chlorine and fluorine, said process com-prising; nitrosolating a compound of the general formula:

with nitrous acid.

2. A process as defined in claim 1, wherein R is neo-pentyl and Hal is chlorine.

3. A process as defined in claim 1, wherein R is neo-hexyl and Hal is chlorine.

4. A process as defined in claim 1, wherein R is iso-pentyl and Hal is chlorine.

5. A process as defined in claim 1, wherein R is iso-butyl and Hal is chlorine.

6. A compound of the general formula:

wherein R is selected from the group consisting of neopentyl, neohexyl, isopentyl and isobutyl and Hal is selected from chlorine and fluorine, whenever prepared by the process of claim 1, or by an obvious chemical equivalent thereof.

7. 1-neopentyl-3-(2-chloroethyl)-3-nitrosourea, whenever prepared by the process of claim 2, or by an obvious chemical equivaleht thereof.

8. 1-neohexyl-3-(2-chloroethyl)-3-nitrosourea, whenever prepared by the process of claim 3, or by an obvious chemical equivalent thereof.

9. 1-isopentyl-3-(2-chloroethyl)-3-nitrosourea, whenever prepared by the process of claim 4, or by an obvious chemical equivalent thereof.

10. 1-isobutyl-3-(2-chloroethyl)-3-nitrosourea, whenever prepared by the process of claim 5, or by an obvious chemical equivalent thereof.

Claims Supported By The Supplementary Disclosure

11. A process for preparing a compound of the general formula:

wherein R is an alkyl group of 3 to 8 carbon atoms with no branching at the Cl atom and Hal is selected from the group consisting of chlorine and fluorine, said process comprising nitrosolating a compound of the general formula:

with nitrous acid.

12. A process as defined in claim 11, wherein said R group is branched at the C2 position.

13. A process as defined in claim 11, wherein said R group is branched at the C3 position.

14. A process as defined in claim 11, wherein said R group sterically conforms to a glutamine residue.

15. A compound of the general formula:
wherein R is an alkyl group of 3 to 8 carbon atoms with no branching at the Cl atom and Hal is selected from chlorine and fluorine, whenever prepared by the process of claim 11, or by an obvious chemical equivalent thereof.

16. A compound of the general formula:

wherein R is an alkyl group of 3 to 8 carbon atoms with no branching at the C1 atom but branched at the C2 atom and Hal is selected from chlorine and fluorine, whenever prepared by the process of claim 12, or by an obvious chemical equivalent thereof.

17. A compound of the general formula:

wherein R is an alkyl group of 3 to 8 carbon atoms with no branching at the Cl atom but branched at the C3 atom and Hal is selected from chlorine and fluorine, whenever prepared by the process of claim 13, or by an obvious chemical equivalent thereof.

18. A compound of the general formula:

wherein R conforms to a glutamine residue and Hal is selected from chlorine and fluorine, whenever prepared by the process of claim 14, or by an obvious chemical equivalent thereof.